Novel Promiscuous HPV16-Derived T Helper Epitopes for Immunotherapy

ABSTRACT

The present invention relates to novel amino acid sequences of peptides derived from HPV 16 that are able to bind to MHC complexes of class II, and elicit an immune response. The present invention further relates to pharmaceutical products, such as vaccines and T-cells, based on said epitopes.

CROSS REFERENCE TO A RELATED APPLICATION

This application is a Divisional Application of co-pending applicationU.S. Ser. No. 15/104,165, filed on Jun. 13, 2016; which is a NationalStage Application of International Application Number PCT/EP2014/076048,filed Dec. 1, 2014; which claims priority to European Application No.13196923.0, filed Dec. 12, 2013; which are incorporated herein byreference in their entirety.

The Sequence Listing for this application is labeled“SeqList-13Jun16.txt”, which was created on Jun. 13, 2016, and is 7 KB.The entire content is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel amino acid sequences of peptidesderived from HPV16 that are able to bind to MHC complexes of class II,and elicit an immune response. The present invention further relates topharmaceutical products, such as vaccines and T-cells, based on saidepitopes.

BACKGROUND OF THE INVENTION

Cervical carcinoma and several other human papillomavirus (HPV)-inducedmalignancies are a global public health problem, thus novel treatmentmodalities are urgently needed. Immunotherapy is an attractive optionfor treatment of infection and (pre)malignant lesions. However, previousapproaches—focusing on the induction of cytotoxic CD8+ T cells—have asyet not yielded substantial clinical successes. Since CD4+ T cells havebeen shown to be crucial for the induction and maintenance of cytotoxicCD8+ T cell responses, and more recently to be also important for directanti-tumor immunity, HLA class II-restricted epitopes are intensivelyinvestigated to improve the efficacy of peptide-based HPV immunotherapy.

High-risk types of human papillomavirus (HPV) are associated withseveral malignant diseases, including cervical carcinoma, otheranogenital tumors, and oropharyngeal carcinomas (zur Hausen H. 1987.Papillomaviruses in human cancer. Appl Pathol 5: 19-24; Bosch F X, etal. The causal relation between human papillomavirus and cervicalcancer. J Clin Pathol 55: 244-65; Gillison M L, et al. 2012. Humanpapillomavirus and diseases of the upper airway: head and neck cancerand respiratory papillomatosis. Vaccine 30 Suppl 5: F34-541-3). Naturalhistory studies indicate that nearly every sexually active individualwill acquire at least one high-risk HPV infection during their lifetime(Baseman J G, Koutsky L A. 2005. The epidemiology of humanpapillomavirus infections. J Clin Virol 32 Suppl 1: S16-24, Schiffman M,Castle P E, Jeronimo J, Rodriguez A C). Fortunately, the majority of HPVinfections are eradicated by the host immune system within 1-2 years ofacquisition (Woodman C B, Collins S I, Young L S. 2007. The naturalhistory of cervical HPV infection: unresolved issues. Nat Rev Cancer 7:11-22, Rodriguez A C, et al. 2010. Longitudinal study of humanpapillomavirus persistence and cervical intraepithelial neoplasia grade2/3: critical role of duration of infection. J Natl Cancer Inst 102:315-24), and only <1% of infected people develop HPV-mediated cancers(Evander M, et al. 1995. Human papillomavirus infection is transient inyoung women: a population-based cohort study. J Infect Dis 171: 1026-30,Koutsky L. 1997. Epidemiology of genital human papillomavirus infection.Am J Med 102: 3-8). Why some individuals clear the viral infection,while others do not, is still incompletely understood. Accumulating datasuggest that both cytotoxic CD8+ T cell (CTL) and CD4+T helper (Th) cellresponses play a pivotal role in the control and clearance of HPVinfection (Stanley M A. 2001. Immunobiology of papillomavirusinfections. J Reprod Immunol 52: 45-59; Welters M J, et al. 2003.Frequent display of human papillomavirus type 16 E6-specific memoryt-Helper cells in the healthy population as witness of previous viralencounter. Cancer Res 63: 636-41; Nakagawa M, et al. 2000. Persistenceof human papillomavirus type 16 infection is associated with lack ofcytotoxic T lymphocyte response to the E6 antigens. J Infect Dis 182:595-8; van der Burg S H, et al. 2002. The status of HPV16-specificT-cell reactivity in health and disease as a guide to HPV vaccinedevelopment. Virus Res 89: 275-84). To date, however, the majority oftherapeutic HPV vaccines have been designed to elicit tumor-specific CTLresponses (Ressing M E, et al. 2000. Detection of T helper responses,but not of human papillomavirus-specific cytotoxic T lymphocyteresponses, after peptide vaccination of patients with cervicalcarcinoma. J Immunother 23: 255-66; Kaufmann A M, et al. 2002. Safetyand immunogenicity of TA-HPV, a recombinant vaccinia virus expressingmodified human papillomavirus (HPV)-16 and HPV-18 E6 and E7 genes, inwomen with progressive cervical cancer. Clin Cancer Res 8: 3676-85;Garcia F, et al. 2004. ZYC101a for treatment of high-grade cervicalintraepithelial neoplasia: a randomized controlled trial. Obstet Gynecol103: 317-26). Unfortunately, only disappointing clinical outcomes havebeen observed, with the exception of one study with synthetic longpeptides in vulvar intraepithelial neoplasia (Kenter G G, et al. 2009.Vaccination against HPV-16 oncoproteins for vulvar intraepithelialneoplasia. N Engl J Med 361: 1838-47). Exclusive targeting of HLA (humanleukocyte antigen) class I-restricted CTL HPV epitopes, withoutinvolving specific T-cell help, can lead to suboptimal and short-lastingCD8+ T cell responses. The lessons learned from these clinical studieslead to a rethinking of therapeutic anti-HPV vaccine design.

Among the eight HPV proteins, E2, E5, E6, and E7 are regarded as beingcrucial for HPV immune escape and malignant progression. E2 and E5 areexpressed soon after infection, prompting HPV immune escape mechanismsand initiating carcinogenic progression (Bellanger S, et al. 2011. Tumorsuppressor or oncogene? A critical role of the human papillomavirus(HPV) E2 protein in cervical cancer progression. Am J Cancer Res 1:373-89; Xue Y, et al. 2012. Loss of HPV16 E2 Protein Expression WithoutDisruption of the E2 ORF Correlates with Carcinogenic Progression. OpenVirol J 6: 163-72; Maufort J P, et al. A role for HPV16 E5 in cervicalcarcinogenesis. Cancer Res 70: 2924-31; Ganguly N. 2012. Humanpapillomavirus-16 E5 protein: oncogenic role and therapeutic value. CellOncol (Dordr) 35: 67-76). E6 and E7 as major transforming proteins areconstitutively expressed in both premalignant and advanced lesions,making them ideal targets for immunotherapeutic approaches forHPV-induced malignancies (zur Hausen H. 2000. Papillomaviruses causingcancer: evasion from host-cell control in early events incarcinogenesis. J Natl Cancer Inst 92: 690-8, Tan S, et al. 2012.Anticancer drugs aimed at E6 and E7 activity in HPV-positive cervicalcancer. Curr Cancer Drug Targets 12: 170-84).

CD4+Th cells are known to play critical roles in the generation of CTLimmune responses as well as memory T cell responses (Wiesel M, OxeniusA. 2012. From crucial to negligible: functional CD8(+) T-cell responsesand their dependence on CD4(+) T-cell help. Eur J Immunol 42: 1080-8;Williams M A, et al. 2006. Developing and maintaining protective CD8+memory T cells. Immunol Rev 211: 146-53). CD4+ T cell help preventspeptide-specific tolerance of CD8+ T cells (Shafer-Weaver K A, et al.2009. Immunity to murine prostatic tumors: continuous provision ofT-cell help prevents CD8 T-cell tolerance and activatestumor-infiltrating dendritic cells. Cancer Res 69: 6256-64). Further,tumor-specific CD4+ T cells may aid recruitment and cytolytic functionof CD8+ T cells in the tumor microenvironment (Bos R, Sherman L A. 2010.CD4+ T-cell help in the tumor milieu is required for recruitment andcytolytic function of CD8+T lymphocytes. Cancer Res 70: 8368-77).Moreover, CD4+ T cells, in absence of CD8+ T cells, may execute directcytotoxic functions in a peptide-specific and HLA class II-restrictedmanner (van de Berg P J, et al. Cytotoxic human CD4(+) T cells. CurrOpin Immunol 20: 339-43; Brown D M. 2010. Cytolytic CD4 cells: Directmediators in infectious disease and malignancy. Cell Immunol 262:89-95). This might be relevant to HPV-induced lesions, as HLA class Isurface expres-expression can be reduced as a result of HPV immuneevasion strategies (Grabowska A K, Riemer A B. 2012. The invisibleenemy—how human papillomaviruses avoid recognition and clearance by thehost immune system. Open Virol J 6: 249-56). In contrast, HLA class IImolecules are expressed in high-grade cervical lesions and cervicalcarcinoma (Coleman N, Stanley M A. 1994. Analysis of HLA-DR expressionon keratinocytes in cervical neoplasia. Int J Cancer 56: 314-9; Zhou JH, et al. 2006. Altered expression of cellular membrane molecules ofHLA-DR, HLA-G and CD99 in cervical intraepithelial neoplasias andinvasive squamous cell carcinoma. Life Sci 78: 2643-9). It has furtherbeen shown that the most effective induction of CTL immune responsesrequires T helper cells recognizing the same cognate antigen, ratherthan an unrelated Th stimulus (Bennett S R, et al. 1997. Induction of aCD8+ cytotoxic T lymphocyte response by cross-priming requires cognateCD4+ T cell help. J. Exp Med 186: 65-70). Therefore, the inventorsbelieve that for rational design of immunotherapeutic approaches againstHPV-mediated lesions is beneficial to identify and provide CD4+T helpercell epitopes derived from HPV target antigens. Most studies onidentification of HPV-specific Th epitopes so far have been focusing onE6 and E7 as target antigens and overlapping peptide pools have beenused rather than defined peptide epitopes (for review see Wang X, et al.A novel CD4 T-cell epitope described from one of the cervical cancerpatients vaccinated with HPV 16 or 18 E7-pulsed dendritic cells. CancerImmunol Immunother 58: 301-8; Welters M J, et al. 2006. Detection ofhuman papillomavirus type 18 E6 and E7-specific CD4+T-helper 1 immunityin relation to health versus disease. Int J Cancer 118: 950-6; Peng S,et al. 2007. HLA-DQB1*02-restricted HPV-16 E7 peptide-specific CD4+T-cell immune responses correlate with regression of HPV-16-associatedhigh-grade squamous intraepithelial lesions. Clin Cancer Res 13:2479-87; van der Burg S H, et al. 2001. Natural T-helper immunityagainst human papillomavirus type 16 (HPV16) E7-derived peptide epitopesin patients with HPV16-positive cervical lesions: identification of 3human leukocyte antigen class II-restricted epitopes. Int J Cancer 91:612-8; Gallagher K M, Man S. 2007. Identification of HLA-DR1- andHLA-DR15-restricted human papillomavirus type 16 (HPV16) and HPV18 E6epitopes recognized by CD4+ T cells from healthy young women. J. GenVirol 88: 1470-8). Although successful, these approaches of systematic Tcell epitope mapping are costly and time-consuming, as they requiresynthesis and several rounds of screening of peptides spanning the fulllength of the target antigen.

U.S. Pat. No. 8,252,893 B2 describes CD8 T cell epitopes in the E6 andE7 protein of Human Papillomavirus (HPV). U.S. Pat. No. 7,026,443 B1discloses human papillomavirus (HPV) epitopes, pharmaceuticalcompositions and methods of use in the prevention and treatment of HPVinfection. EP2167137A1 discloses HPV polyepitope constructs and the usethereof for the prevention and/or treatment of HPV infection.

The identification and characterization of HPV-associated antigensrecognized by CD4-positive T cells (ligand: MHC class IImolecule+peptide epitope) is important for the further development ofHPV vaccines. It is therefore an object of the present invention, toprovide novel amino acid sequences for HPV-derived peptides that areable to bind to MHC complexes of class II.

In a first aspect thereof, the invention provides a peptide comprising asequence selected from the group of SEQ ID Nos. 26, 17, 28, 30, 1 to 16,18 to 25, 27, 29, and 31 or a variant thereof which is 80% homologous,preferably 90%, and most preferred 95% homologous to any of SEQ ID Nos.26, 17, 28, 30, 1 to 16, 18 to 25, 27, 29, and 31, wherein said peptideor variant thereof has the ability to bind to a molecule of the humanmajor histocompatibility complex (MHC) class-II. In other variants, oneor two amino acids at the N- and/or C-terminal end of a sequenceselected from the group of SEQ ID Nos. 26, 17, 28, 30, 1 to 16, 18 to25, 27, 29, and 31 can be replaced with another amino acid (preferablyhaving the same or similar characteristics), wherein said variantmaintains the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-II, preferably the same as theunderlying sequence selected from the group of SEQ ID Nos. 26, 17, 28,30, 1 to 16, 18 to 25, 27, 29, and 31 (see also below).

In the present invention, the term “homologous” refers to the degree ofidentity between sequences of two amino acid sequences, i.e. peptide orpolypeptide sequences. The aforementioned “homology” is determined bycomparing two sequences aligned under optimal conditions over thesequences to be compared. The sequences to be compared herein may havean addition or deletion (for example, gap and the like) in the optimumalignment of the two sequences. Such a sequence homology can becalculated by creating an alignment using, for example, the ClustalWalgorithm (Nucleic Acid Res., 22(22): 4673 4680 (1994)). Commonlyavailable sequence analysis software, more specifically, Vector NTI,GENETYX or analysis tools provided by public databases, such as e.g.http://dragon.bio.purdue.edu/bioinfolinks/ may also be used.

Table A shows the peptides according to the invention, and theirrespective SEQ ID NOs.

SEQ ID NO: Peptide Sequence  1 PRKLPQLCTELQTTI  2 QQLLRREVYDFAFRD  3FRDLCIVYRDGNPYA  4 LKFYSKISEYRHYCY  5 GTTLEQQYNKPLCDL  6 KQRFHNIRGRWTGRC 7 MLDLQPETTDLYCYE  8 STLRLCVQSTHVDIR  9 TLRLCVQSTHVDIRT 10STHVDIRTLEDLLMG 11 HVDIRTLEDLLMGTL 12 IRTLEDLLMGTLGIV 13 RDHIDYWKHMRLECA14 FKHINHQVVPTLAVS 15 VPTLAVSKNKALQAI 16 QLTLETIYNSQYSNE 17LEVYLTAPTGCIKKH 18 GLYYVHEGIRTYFVQ 19 PEIIRQHLANHPAAT 20 ILTAFNSSHKGRINC21 IVHLKGDANTLKCLR 22 CLRYRFKKHCTLYTA 23 DQFLSQVKIPKTITV 24MTNLDTASTTLLACF 25 RPLLLSVSTYTSLIL 26 LLSVSTYTSLILLVL 27 LVLVLWITAASAFRC28 ASAFRCFIVYIVFVY 29 FIVYIVFVYIPLFLI 30 VYIPLFLIHTHARFL 31IPLFLIHTHARFLITPreferred are the following sequences (table A1):

SEQ ID NO: Peptide Sequence  3 FRDLCIVYRDGNPYA  4 LKFYSKISEYRHYCY  7MLDLQPETTDLYCYE  9 TLRLCVQSTHVDIRT 12 IRTLEDLLMGTLGIV 17 LEVYLTAPTGCIKKH18 GLYYVHEGIRTYFVQ 26 LLSVSTYTSLILLVL 28 ASAFRCFIVYIVFVY 30VYIPLFLIHTHARFL

Most preferred are the following sequences (table A2):

SEQ ID NO: Peptide Sequence  4 LKFYSKISEYRHYCY  7 MLDLQPETTDLYCYE  9TLRLCVQSTHVDIRT 18 GLYYVHEGIRTYFVQ 26 LLSVSTYTSLILLVL

Thus, the invention also relates to a combination of peptides accordingto the invention, comprising at least two, preferably three, four orfive peptides consisting of an amino acid sequence selected from thegroup of SEQ ID Nos. 26, 9, 7, 4, and 18, in particular in the form of avaccine such as, for example, a cancer vaccine.

In the present study, with the help of immuno-bioinformatic approaches,the inventors identified several candidate HPV16 E2-, E5-, E6-, andE7-derived CD4+ T cell epitopes promiscuously presented by multipleHLA-DR alleles and evaluated their immunogenicity in healthyindividuals. The inventors here present an approach to identifypromiscuous HPV16-derived CD4+T helper epitopes, which are able toinduce T cell immunity in a large proportion of the population. To thisend, the inventors combined HLA class II epitope prediction servers withex vivo immunological evaluation to identify HPV 16 E2-, E5-, E6-, andE7-derived CD4+ T cell epitopes. Candidate selected HPV16-derivedepitopes were found to be restricted by up to nine out of eleven testedHLA-DR molecules. Furthermore, they were found to induce frequent androbust HPV16 peptide-specific T cell responses in the healthy donors, asmonitored by IFN-γ ELISPOT and cytokine secretion assays. The observedHPV16 peptide-specific immunity was of the Th1 phenotype, and notassociated with CD4+ regulatory T cells. Therefore, the inventorsconclude that the identified T helper epitopes are valuable candidatesfor the development of a comprehensive therapeutic HPV vaccine.

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)so that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence. For example, a peptide may be modified so that it atleast maintains, if not improves, the ability to interact with and binda suitable MHC molecule, such as HLA-DR, and so that it at leastmaintains, if not improves, the ability to generate activated CTL thatcan recognize and kill cells that express a polypeptide containing anamino acid sequence as defined in the aspects of the invention.

Those amino acid residues that are not essential to interact with theT-cell receptor can be modified by replacement with another amino acidwhose incorporation does not substantially affect T-cell reactivity anddoes not eliminate binding to the relevant MHC. Thus, apart from theproviso given, the peptide of the invention may be any peptide (by whichterm the inventors include oligopeptide or polypeptide) which includesthe amino acid sequences or a portion or variant thereof as given.

It is furthermore known for MHC-class II presented peptides that thesepeptides are composed of a “core sequence” having a certain HLA-specificamino acid motif and, optionally, N- and/or C-terminal extensions whichdo not interfere with the function of the core sequence (i.e. are deemedirrelevant for the interaction of the peptide and all or a subset ofT-cell clones recognizing the natural counterpart). The N- and/orC-terminal extensions can, for example, be between 1 to 10 amino acidsin length, respectively. These peptides can be used either directly toload MHC class II molecules or the sequence can be cloned into thevectors according to the description herein below. As these peptidesconstitute the final product of the processing of larger peptides withinthe cell, longer peptides can be used as well. The peptides of theinvention may be of any size, but typically they may be less than 5.000in molecular weight. In terms of the number of amino acid residues, thepeptides of the invention may have fewer than 50 residues. Accordinglythe present invention also provides peptides and variants thereofwherein the peptide or variant has an overall length of between 15 and50, preferably between 15 and 30, and most preferred between 15 and 20amino acids, namely 15, 16, 17, 18, 19, or 20 amino acids Binding of apeptide or a variant to a MHC complex may be tested by methods known inthe art, for example those described in example 4 of the presentinvention or those described in the literature for different MHC classII alleles (e.g. Vogt A B, et al. Ligand motifs of HLA-DRB5*0101 andDRB1*1501 molecules delineated from self-peptides; J Immunol. 1994;153(4):1665-1673; Malcherek G, et al. Analysis of allele-specificcontact sites of natural HLA-DR17 ligands; J Immunol. 1994;153(3):1141-1149; Manici S, et al. Melanoma cells present a MAGE-3epitope to CD4(+) cytotoxic T cells in association withhistocompatibility leukocyte antigen DR11; J Exp Med. 1999; 189(5):871-876; Hammer J, et al. Peptide binding specificity of HLA-DR4molecules: correlation with rheumatoid arthritis association; J Exp Med.1995 181(5):1847-1855; Tompkins S M, Rota P A, Moore J C, Jensen P E; Aeuropium fluoroimmunoassay for measuring binding of antigen to class IIMHC glycoproteins; J Immunol Methods. 1993; 163(2): 209-216; Boyton R J,et al. Glutamic acid decarboxylase T lymphocyte responses associatedwith susceptibility or resistance to type I diabetes: analysis indisease discordant human twins, non-obese diabetic mice and HLA-DQtransgenic mice; Int Immunol. 1998 (12):1765-1776).

The peptides of the present invention or variants thereof are expectedto stimulate CD4+ T cells.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID Nos. 26, 17, 28, 30, 1 to 16, 18 to 25, 27, 29, and 31.“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID Nos. 26, 17, 28, 30, 1 to 16, 18 to 25, 27, 29, and 31 or a variantthereof, contains additional N- and/or C-terminally located stretches ofamino acids that are not necessarily forming part of the peptide thatfunctions as an epitope for MHC molecules epitope.

In a particularly preferred embodiment of the invention, the peptide ofthe invention has an IC₅₀: 60≤1000 nM, and preferably an IC₅₀≤60 nM foran HLA-DR allele.

In addition the peptide or variant may be modified further to improvestability and/or binding to MHC molecules to elicit a stronger immuneresponse. Methods for such an optimization of a peptide sequence arewell known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains that involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2005, which is incorporatedherein by reference. Chemical modification of amino acids includes butis not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley & Sons NY 1995-2000) for more extensivemethodology relating to chemical modification of proteins.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Luet al J. Org. Chem. 1981, 46, 3433, and references therein, as well asthrough other methods known in the art. Purification may be effected byany one, or a combination of, techniques such as re-crystallization,size exclusion chromatography, ion-exchange chromatography, hydrophobicinteraction chromatography and (usually) reverse-phase high performanceliquid chromatography using e.g. acetonitril/water gradient separation.

A further aspect of the invention provides a nucleic acid (e.g.polynucleotide) encoding a peptide or variant of the invention. Thepolynucleotide may be e.g. DNA, cDNA, PNA, CNA, RNA, mRNA, and siRNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilised forms of polynucleotides, such as e.g. polynucleotideswith a phosphorothiate backbone, and it may or may not contain intronsso long as it codes for the peptide. Of course, only peptides containingnaturally occurring amino acid residues joined by naturally occurringpeptide bonds are encodable by a polynucleotide. A still further aspectof the invention provides an expression vector capable of expressing apolypeptide according to the invention. A variety of methods have beendeveloped to operably link polynucleotides, especially DNA, to vectorsfor example via complementary cohesive termini. For instance,complementary homopolymer tracts can be added to the DNA segment to beinserted to the vector DNA. The vector and DNA segment are then joinedby hydrogen bonding between the complementary homopolymeric tails toform recombinant DNA molecules.

If viral vectors are used, herpes-, pox- or adenovirus vectors arepreferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention.

Thus, the DNA encoding the peptide or variant of the invention may beused in accordance with known techniques, appropriately modified in viewof the teachings contained herein, to construct an expression vector,which is then used to transform an appropriate host cell for theexpression and production of the polypeptide of the invention.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

In another embodiment the peptide according to the invention, thenucleic acid according to the invention or the expression vectoraccording to the invention are used in medicine. For example, thepeptide or its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramus-cular (i.m.) injection.Preferred routes of peptide injection are s.c., i.d., i.p., i.m., andi.v. Preferred routes of DNA injection are i.d., i.m., s.c., i.p. andi.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μg to 500μg, of peptide or DNA may be given and will depend from the respectivepeptide or DNA.

Preferably, the peptides of the invention are produced synthetically, orat least in part synthetically, using well-established peptide synthesistechnology.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellthus comprises the nucleic acid according to the invention or theexpression vector according to the invention. The host cell can beeither prokaryotic or eukaryotic. Bacterial cells may be preferredprokaryotic host cells in some circumstances and typically are a strainof E. coli such as, for example, the E. coli strains DH5 available fromBethesda Research Laboratories Inc., Bethesda, Md., USA, and RR1available from the American Type Culture Collection (ATCC) of Rockville,Md., USA (No ATCC 31343). Preferred eukaryotic host cells include yeast,insect and mammalian cells, preferably vertebrate cells such as thosefrom a mouse, rat, monkey or human. Yeast host cells include YPH499,YPH500 and YPH501 which are generally available from Stratagene CloningSystems, La Jolla, Calif. 92037, USA. Preferred mammalian host cellsinclude Chinese hamster ovary (CHO) cells available from the ATCC asCCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC asCRL 1658, monkey kidney-derived COS-1 cells available from the ATCC asCRL 1650 and 293 cells which are human embryonic kidney cells. Preferredinsect cells are Sf9 cells which can be transfected with baculovirusexpression vectors. Transformation of appropriate cell hosts with a DNAconstruct of the present invention is accomplished by well-known methodsthat typically depend on the type of vector used.

A further aspect of the invention provides a method of producing apeptide or its variant. The method comprises culturing the host cell ofthe invention, and isolating the peptide from the host cell or theculture medium.

Another aspect of the present invention relates to a vaccine comprisingthe host cells as described herein, which in a preferred embodiment is alife vaccine. In such an embodiment, the host cell can be an antigenpresenting cell, in particular a dendritic cell. APCs loaded with arecombinant fusion protein containing prostatic acid phosphatase (PAP)are approved for the treatment of prostate cancer (Sipuleucel-T) (see,e.g., Cheever M A, Higano C S. PROVENGE (Sipuleucel-T) in prostatecancer: the first FDA-approved therapeutic cancer vaccine. Clin CancerRes. 2011 Jun. 1; 17(11):3520-6; Epub 2011 Apr. 6).

Another aspect of the present invention is an in vitro method forinducing activated T helper (T_(H)) cells, comprising contacting invitro T_(H) cells with antigen loaded human class II MHC moleculesexpressed on the surface of a suitable antigen-presenting cell for aperiod of time sufficient to activate said T_(H) cells in an antigenspecific manner, wherein said antigen is a peptide according to theinvention. Preferably, a sufficient amount of the antigen is used withan antigen-presenting cell.

The MHC class II molecules may be expressed on the surface of anysuitable cell. Thus, the antigen-presenting cell (or stimulator cell)typically has MHC class II molecules on its surface. The MHC class IImolecule may readily be loaded with the selected antigen in vitro. Incase of MHC II epitopes as antigens, the T cells are CD4-positiveT-helper cells.

A number of methods for generating activated T cells in vitro aredescribed in the respective literature.

The activated T_(H) cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T_(H) cells, produced and obtainable by the methodaccording to the invention, which selectively recognizes a cell whichexpresses an HPV polypeptide comprising an amino acid sequence accordingto SEQ ID Nos. 26, 17, 28, 30, 1 to 16, 18 to 25, 27, 29, or 31.

The T_(H) cells of the invention may be used as active ingredients in atherapeutic composition. Thus the invention also provides a method ofkilling target cells in a patient where the target cells express apolypeptide comprising an amino acid sequence of the invention. Themethod comprises administering to the patient an effective number ofT_(H) cells as defined above.

T_(H) cells may be obtained by methods known in the art, e.g. thosedescribed above.

Any molecule of the invention, i.e. the peptide, nucleic acid,expression vector, cell, activated T_(H) cells, T-cell receptor or thenucleic acid encoding it is useful for the treatment of HPV-relateddisorders, characterized by cells escaping an immune response. Thereforeany molecule of the present invention may be used as medicament or inthe manufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

An important aspect of the present invention is therefore apharmaceutical preparation, comprising at least one of the peptideaccording to the invention, the nucleic acid according to the invention,the expression vector according to the invention, the host cellaccording to the invention, the activated T_(H) lymphocyte according tothe invention, and a pharmaceutically acceptable excipient, whereinpreferably said pharmaceutical preparation is a vaccine.

Preferably the medicament is a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically, or applied exvivo to cells derived from the patient or a human cell line which aresubsequently administered to the patient, or used in vitro to select asubpopulation from immune cells derived from the patient, which are thenre-administered to the patient. If the nucleic acid is administered tocells in vitro, it may be useful for the cells to be transfected so asto co-express immune-stimulating cytokines, such as interleukin-2. Thepeptide may be substantially pure, or combined with animmune-stimulating adjuvant (see below) or used in combination withimmune-stimulatory cytokines, or be administered with a suitabledelivery system, for example nanoparticles or liposomes. The peptide mayalso be conjugated to a suitable carrier such as keyhole limpethaemocyanin (KLH) or mannan (see WO 95/18145 and Longenecker et al.(1993) Ann. NY Acad. Sci. 690, 276-291).

Thus, in another aspect of the invention, the peptide according to theinvention is part of a fusion protein or fusion molecule, e.g. with aTLR ligand.

In one aspect of the invention, the vaccine comprises at least onepeptide, preferably two to 31, more preferably two to 25, even morepreferably two to 15 and most preferably two, three, four, five, six,seven, eight, nine, ten, eleven, twelve or thirteen peptides of theinvention or additional peptides. The peptide(s) may be derived from oneor more specific HPV antigen, and may bind to MHC class I and/or class11 molecules.

Particularly preferred vaccines include peptides according to thepresent invention that show an IC₅₀≤60 nM for an HLA-DR allele.

More preferred is an HPV vaccine comprising at least one, preferablyall, of the following peptides (table A3):

SEQ ID NO: Peptide Sequence  3 FRDLCIVYRDGNPYA  4 LKFYSKISEYRHYCY  7MLDLQPETTDLYCYE  9 TLRLCVQSTHVDIRT 12 IRTLEDLLMGTLGIV 17 LEVYLTAPTGCIKKH18 GLYYVHEGIRTYFVQ 26 LLSVSTYTSLILLVL 28 ASAFRCFIVYIVFVY 30VYIPLFLIHTHARFL

Most preferred is an HPV vaccine comprising at least one, preferablyall, of the following peptides (table A4):

SEQ ID NO: Peptide Sequence  4 LKFYSKISEYRHYCY  7 MLDLQPETTDLYCYE  9TLRLCVQSTHVDIRT 18 GLYYVHEGIRTYFVQ 26 LLSVSTYTSLILLVL

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g. immune responses mediated by CTLs and helper-T(T_(H)) cells to an antigen, and would thus be considered useful in themedicament of the present invention.

Suitable adjuvants include, but are not limited to aluminum salts, TLRagonists, or GM-CSF or other commercial adjuvants.

When the peptides of the invention are used in a vaccine or medicamentof the invention, they are present as a salt, such as for example, butnot limited to an acetate salt or a chloride salt.

Another aspect of the invention then relates to the peptide according tothe invention, the nucleic acid according to the invention, theexpression vector according to the invention, the host cell according tothe invention, the activated T_(H) cell according to the invention, orthe pharmaceutical preparation according to the invention for use in thetreatment of HPV infection, and HPV-related pre-malignancies and/ormalignancies.

Yet another aspect of the invention relates to a method of killingtarget cells in a patient which target cells express an HPV polypeptidecomprising an amino acid sequence as given for the peptides herein, themethod comprising administering to the patient an effective number ofT_(H) cell according to the invention.

In yet another aspect thereof, the present invention relates to a kitcomprising (a) a container that contains a pharmaceutical composition asdescribed above, in solution or in lyophilized form; (b) optionally, asecond container containing a diluent or reconstituting solution for thelyophilized formulation; and (c) optionally, instructions for (i) use ofthe solution or (ii) reconstitution and/or use of the lyophilizedformulation. Said kit may further comprise one or more of (iii) abuffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe.The container is preferably a bottle, a vial, a syringe or test tube;and it may be a multi-use container. The pharmaceutical composition ispreferably lyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contains instructions on or associated with thecontainer that indicates directions for reconstitution and/or use. Forexample, the label may indicate that the lyophilized formulation is toreconstituted to peptide concentrations as described above.

Numerous reports demonstrate the importance of both CD8+ and CD4+ Tcells in the clearance of HPV infection as well as in the protectionagainst developing HPV-associated malignancies (10-13). However, thedevelopment of successful anti-HPV immunotherapy has proved to be achallenging task. Recently, there has been considerable interest toidentify HPV E6 and E7-derived CD4+T helper cell epitopes usingoverlapping peptide pools and to include them in therapeuticpeptide-based therapeutic vaccine design (34-38). The inventors'approach in the current work was to use computational HLA class IIprediction algorithms to define a 15-mer HPV16-derived CD4+ T cellepitopes that bind promiscuously to the binding groove of multipleHLA-DR molecules. The inventors performed predictions for elevenalleles, namely DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0901,DRB1*1101, DRB1*1302, DRB1*1501, DRB3*0011, DRB4*0101 and DRB5*0101.Candidate promiscuous epitopes were evaluated in vitro for theirpotential to elicit immune responses in healthy subjects. As mostsexually active individuals are infected transiently with at least onehigh-risk HPV type during their life (Woodman C B, Collins S I, Young LS. 2007. The natural history of cervical HPV infection: unresolvedissues. Nat Rev Cancer 7: 11-22, Evander M, et al. 1995. Humanpapillomavirus infection is transient in young women: a population-basedcohort study. J. Infect Dis 171: 1026-30), previous HPV exposure of thedonors is likely.

The use of prediction servers has several advantages over usingoverlapping peptide pools. Testing overlapping epitopes spanning thefull length of a protein is a time-consuming approach even when testingonly one HLA type, but even more so when several HLA molecules have tobe interrogated for the identification of promiscuously bindingepitopes. Thus, in the present study, the inventors took advantage oftwo HLA class II epitope prediction servers, SYFPEITHI and NetMHCII.This allowed them to screen in silico for candidate peptides binding toseveral HLA class II molecules. Only peptides predicted to bind to fiveor more HLA-DR molecules were further analyzed, thus focusing immuneassays to the most promising candidates. As the accuracy of predictionservers is not absolute, it is recommendable to use more than one, toensure that no potential binder is missed. Also in this study, it can beseen that the results from the two prediction servers were notcompletely consistent. The combined use of both let to the inclusion ofpeptides that would not have been found by applying only one predictionalgorithm.

Up to now, most studies focused on the identification of epitopesderived from the HPV oncoproteins E6 and E7, in the context ofpeptide-based immunotherapy against advanced lesions in the patients(Wang X, et al. 2009. A novel CD4 T-cell epitope described from one ofthe cervical cancer patients vaccinated with HPV 16 or 18 E7-pulseddendritic cells. Cancer Immunol Immunother 58: 301-8; Welters M J, etal. 2006. Detection of human papillomavirus type 18 E6 and E7-specificCD4+T-helper 1 immunity in relation to health versus disease. Int JCancer 118: 950-6; Peng S, et al. 2007. HLA-DQB1*02-restricted HPV-16 E7peptide-specific CD4+ T-cell immune responses correlate with regressionof HPV-16-associated high-grade squamous intraepithelial lesions. ClinCancer Res 13: 2479-87; van der Burg S H, et al. 2001. Natural T-helperimmunity against human papillomavirus type 16 (HPV16) E7-derived peptideepitopes in patients with HPV16-positive cervical lesions:identification of 3 human leukocyte antigen class II-restrictedepitopes. Int J Cancer 91: 612-8; Gallagher K M, Man S. 2007.Identification of HLA-DR1- and HLA-DR15-restricted human papillomavirustype 16 (HPV16) and HPV18 E6 epitopes recognized by CD4+ T cells fromhealthy young women. J Gen Virol 88: 1470-8). To the best of theinventors' knowledge, there is only one study reporting CD4+ T cellepitopes for HPV16 E2 (de Jong A, et al. 2002. Frequent detection ofhuman papillomavirus 16 E2-specific T-helper immunity in healthysubjects. Cancer Res 62: 472-9), and no studies on E5-derived T helperepitopes. None of these studies deal with the identification ofpromiscuous CD4+ T cell epitopes. As the inclusion of such epitopes inany peptide-based therapeutic HPV vaccine is expected to increaseimmunogenicity and efficacy, the inventors here aimed to identifypromiscuous T helper epitopes derived from HPV16 E2, E5, E6 and E7.

Overall, the inventors found that 13 out of the 31 predictedpromiscuously binding HPV16-derived HLA class II peptides (Table 1) wererecognized by at least 30% of healthy donors in short-term ex vivoELISPOT assays (Tables 2 and 3). The inventors identified seven novelHPV16 15-mer CD4+ T cell epitopes, namely E2/76-90, E5/33-48, E5/45-60,E5/54-69, E5/60-74, E5/67-81 and E7/12-26. The epitopes E6/54-68,E6/74-88, E7/64-78, E7/71-85, E2/99-113 and E2/156-170 are part ofpreviously reported long peptides (Welters M J, et al. 2003. Frequentdisplay of human papillomavirus type 16 E6-specific memory t-Helpercells in the healthy population as witness of previous viral encounter.Cancer Res 63: 636-41, Wang X, et al. A novel CD4 T-cell epitopedescribed from one of the cervical cancer patients vaccinated with HPV16 or 18 E7-pulsed dendritic cells. Cancer Immunol Immunother 58: 301-8,Peng S, et al. 2007. HLA-DQB1*02-restricted HPV-16 E7 peptide-specificCD4+ T-cell immune responses correlate with regression ofHPV-16-associated high-grade squamous intraepithelial lesions. ClinCancer Res 13: 2479-87, de Jong A, et al. 2002. Frequent detection ofhuman papillomavirus 16 E2-specific T-helper immunity in healthysubjects. Cancer Res 62: 472-9), supporting the inventors' approach ofepitope detection. Each peptide was predicted to bind to five to ninedifferent HLA class II molecules out of the eleven tested in this study,and found to elicit immune responses in individuals carrying therespective HLA class II alleles (Table 4). Only three subjects showedreactivity towards a peptide that was not predicted to bind to any ofhis/her HLA-DR molecules. This reactivity could be due to HLA-DP or -DQpeptide presentation, or HLA class I restricted epitopes contained inthe respective 15-mers (see below). Another interesting finding was thehigh proportion of HPV16 E7-specific CD4+ T cell responses in thecohort, although it has been reported that E7-reactivity is rarelyobserved in healthy individuals (Welters M J, et al. 2003. Frequentdisplay of human papillomavirus type 16 E6-specific memory t-Helpercells in the healthy population as witness of previous viral encounter.Cancer Res 63: 636-41 van der Burg S H, et al. 2001. Natural T-helperimmunity against human papillomavirus type 16 (HPV 16) E7-derivedpeptide epitopes in patients with HPV 16-positive cervical lesions:identification of 3 human leukocyte antigen class II-restrictedepitopes. Int J Cancer 91: 612-8; Gallagher K M, Man S. 2007.Identification of HLA-DR1- and HLA-DR15-restricted human papillomavirustype 16 (HPV16) and HPV18 E6 epitopes recognized by CD4+ T cells fromhealthy young women. J Gen Virol 88: 1470-8).

Based on ELISPOT reactivities (FIG. 1), five epitopes were selected fordetailed analysis. HLA class II restriction of the HPV16peptide-specific T cell responses was confirmed by abrogation of IFN-γproduction in the presence of an HLA-DR blocking antibody (FIG. 2).Possible HLA-DQ and HLA-DP restriction of the selected peptides was notassessed in the inventors' study. Regarding the phenotype of HPV16peptide-specific T cell immunity, the inventors' results from CD4+ andCD8+ T cell depletion (FIG. 3) and intracellular cytokine stainingexperiments provide strong evidence that the responding T cells areindeed CD4+ T cells. The profile of cytokine secretion of HPV16peptide-specific CD4+T cells was found to be of the Th1 type (IFN-γ,TNF-α, GM-CSF) (FIG. 4A), which is asso-associated with control andclearance of HPV infections (Scott M, Stites D P, Moscicki A B. 1999.Th1 cytokine patterns in cervical human papillomavirus infection. ClinDiagn Lab Immunol 6: 751-5; Bais A G, et al. 2007. Cytokine release inHR-HPV(+) women without and with cervical dysplasia (CIN II and III) orcarcinoma, compared with HR-HPV(−) controls. Mediators Inflamm 2007:24147). Moreover, the inventors observed only low percentages of HPV16peptide-specific CD4+ regulatory T cells (FIG. 4B and FIG. 5), which isalso associated with a favorable disease outcome (van der Burg S H, etal. 2007. Association of cervical cancer with the presence of CD4+regulatory T cells specific for human papillomavirus antigens. Proc NatlAcad Sci USA 104: 12087-92; Kim K H, et al. 2012. CD4+ T-cell responseagainst human papillomavirus type 16 E6 protein is associated with afavorable clinical trend. Cancer Immunol Immunother 61: 63-70; Visser J,et al. 2007. Frequencies and role of regulatory T cells in patients with(pre)malignant cervical neoplasia. Clin Exp Immunol 150: 199-209). Asthe inventors tested healthy donors who are likely to have cleared HPVinfection in their past, these results are promising that the inventors'strategy indeed identifies peptides involved in viral clearance.

CD4+ T cells have recently been reported to be capable of directcytotoxic functions (van de Berg P J, et al. 2008. Cytotoxic humanCD4(+) T cells. Curr Opin Immunol 20: 339-43; Brown D M. 2010. CytolyticCD4 cells: Direct mediators in infectious disease and malignancy. CellImmunol 262: 89-95), therefore perforin/granzyme B release as well asCD107a expression analyses were performed. However, no HPV16-derivedpeptide-specific CD4+ T cell-mediated cytotoxic activity was observed inthis cohort of healthy individuals (data not shown). This observationmight reflect the fact that cytotoxic activity of T helper cells arisesonly during acute or persistent infections or in the presence ofmalignancies (Brown D M. 2010. Cytolytic CD4 cells: Direct mediators ininfectious disease and malignancy. Cell Immunol 262: 89-95).

Importantly, it is possible that defined CD4+ T cell epitopes harbor HLAclass I-restricted immunogenic regions. HLA class II-restricted epitopesthat contain HLA class I epitopes in their sequence could be highlyuseful for effective peptide-based immunotherapies, as both CD4+ andCD8+ T cell responses may be simultaneously induced.

In summary, the inventors have defined 13 promiscuous 15-merHPV16-derived CD4+ T cell epitopes, seven of which have not beendescribed before. The inventors show that selected epitopes indeed arepromiscuous and immunogenic, inducing Th1 cells in healthy individualswith a diverse genetic background. The inventors conclude that theidentified T helper epitopes are candidates for the development of acomprehensive epitope-specific therapeutic HPV vaccine againstestablished premalignant as well as advanced lesions.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described in more detail in the followingexamples with reference to the accompanying Figures and the sequencelisting, nevertheless, without being limited thereto. For the purposesof the present invention, all references as cited herein areincorporated by reference in their entireties. In the Figures,

FIG. 1 shows the detection of HPV16 peptide-specific T cell immunity inhealthy subjects. The PBMC reactivity of 13 healthy donors was evaluatedby stimulating with selected HPV16-derived peptides for 12 days. IFN-γpeptide-specific T cells were analyzed by ELISPOT. Each dot representsthe number of positive cells in one individual. Data represent the meanfrom triplicate wells after background subtraction. Mean responses (±SE)across donors are shown for each peptide. Responses are consideredpositive (●) if they were at least twofold higher than the background.An open circle (∘) represents a non-responder. Percentages representresponse rate of tested healthy subjects to the indicated HPV16-derivedpeptide.

FIGS. 2A-2B show that T cell immunity induced by the selectedHPV16-derived peptides is dependent on HLA class II peptidepresentation. PBMC obtained from subject HD54 (2A) and HD55 (2B) wereincubated in the presence or absence of anti-HLA-DR or panHLA-I W6/32antibodies prior the indicated peptide exposure, and responses analyzedby IFN-γ ELISPOT. Data represent the mean result from triplicate wellsafter background subtraction. Significant inhibition in presence ofHLA-DR antibodies compared to non-treated PBMC is indicated by *(P≤0.05, two-tailed Student's t-test). The HLA genotypes of testedsubjects are indicated above the panels.

FIGS. 3A-3C show that the induced HPV16 peptide-specific T cell immunityis mediated by CD4+ T cells. PBMC of 7 healthy donors after stimulationwith the indicated HPV16-derived peptides were tested by flowcytometry-based IFN-γ intracellular staining (ICS) (3A). Each dotrepresents an individual. Results are presented as percentage ofIFN-γ-positive CD4+(●) or CD8+(∘) cells after background subtraction.(3B) PBMC (●) obtained from 12 healthy individuals were depleted of CD4+T cells (∘), stimulated with selected peptides, and analyzed by IFN-γELISPOT. Each dot represents an individual. Data represent the meanresult from triplicate wells after background subtraction. (3C) PBMCs oftwo subjects (HD23 and HD24) were depleted of either CD4+ or CD8+ Tcells prior to peptide exposure as indicated and analyzed by IFN-γELISPOT. Data represent the mean result from triplicate wells afterbackground subtraction. The HLA genotypes of tested subjects areindicated above the panels. Mean responses (±SE) are indicated for eachpeptide. Significant differences are indicated by * (P≤0.05), **(P≤0.01) or *** (P≤0.001, two-tailed Student's t-test).

FIGS. 41-4B show that the induced HPV16 peptide-specific immunity isassociated with Th1-type cells but not with CD4+ regulatory T cells.(4A) The supernatants of T cell cultures from HD23 were tested 3 daysafter in vitro stimulation with indicated peptides for the indicatedcytokines. Data represent the mean result from duplicate wells afterbackground subtraction. The cut-off for positivity is indicated by adashed line. (4B) PBMC were also investigated for presence ofCD4+/CD25+/FoxP3+ regulatory T cells after 8 day in vitro stimulationwith indicated peptides or without stimulation (DMSO) in a flowcytometry-based assay. Results are presented as percentage ofCD25+/FoxP3+ T cells on the counter plot gated on CD4+ T cells.Representative results of one donor (HD 23) are shown. The HLA genotypeof the tested subject is indicated above the panel.

FIG. 5 shows that PBMC of five healthy donors were investigated forpresence of CD4+/CD25+/FoxP3+ regulatory T cells after 12 days in vitrostimulation with indicated peptides or without stimulation (DMSO) in aflow cytometry-based assay. Results are presented as percentage ofCD25+/FoxP3+ T cells. Each dot represents an individual. Mean responses(+SE) across donors are shown for each peptide.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NOs: 1 to 31 shows peptide sequences of HPV-related antigens andepitopes according to the invention.

DETAILED DESCRIPTION OF THE INVENTION Examples Materials and MethodsPrediction of Promiscuous HLA Class II-Binding HPV-Derived Epitopes

An epitope prediction server, SYFPEITHI (see Worldwide Web site:syfpeithi.com) (39) was used for predicting 15-mer Th epitopes in E7(GenBank: AAD33253.1), E6 (GenBank: AAD33252.1), E5 (GenBank:AA085413.1), and E2 (GenBank: AAD33255.1) proteins of HPV 16. The insilico predictions were performed for multiple HLA-DR molecules encodedby DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101 and DRB1*1501alleles. Higher binding score values indicate higher predicted bindingaffinity of candidate peptides to the selected HLA-DR allele. Toidentify promiscuous CD4+ T cell epitopes, for each protein, candidatepeptides were ranked according to their predicted binding affinity amongthe indicated HLA-DR alleles, excluding epitopes harboring more than twocystein residues. Peptides predicted to bind at least five HLA-DRmolecules were selected. In addition, HLA-DR binding epitopes wereanalyzed for dataset cross-comparison by the recently developed NetMHCII(NN-align) prediction method (Nielsen M, et al. 2010.NetMHCIIpan-2.0—Improved pan-specific HLA-DR predictions using a novelconcurrent alignment and weight optimization training procedure.Immunome Res 6: 9, Nielsen M, Lund O. 2009. NN-align. An artificialneural network-based alignment algorithm for MHC class II peptidebinding prediction. BMC Bioinformatics 10: 296) (see Worldwide Website:cbs.dtu.dk/services/NetMHCII/), which also allows to perform predictionsfor molecules encoded by DRB1*0901, DRB1*1302, DRB3*0101, DRB4*0101 andDRB5*0101 alleles. In this case, lower IC₅₀ nM values indicate higherbinding affinity of peptides to a HLA-DR molecule.

Peptides

The 15-mer peptides were synthesized using 9-fluorenylmethoxycarbonylchemistry, and purified by high-performance liquid chromatography (>95%purity) and validated by mass spectrometry (Genomics and Proteomics CoreFacility, German Cancer Research Center (DKFZ), Heidelberg, Germany).Synthetic peptides were reconstituted in DMSO (Sigma, Taufkirchen,Germany) at a concentration of 10 mg/ml, and stored at −80° C. A MHCclass II peptide pool (PANATecs, Tibingen, Germany) was included inexperiments as a positive control.

Healthy Volunteers and Peripheral Blood T Cell Sample Acquisition

Peripheral blood samples or buffy coats of whole blood from anonymoushealthy individuals were obtained from the IKTZ Heidelberg Blood Bank.As these donors are anonymous, no data are available to determine HPVinfection status. Peripheral blood mononuclear cells (PBMC) wereisolated within 12 h using a standard Ficoll-Hypaque density gradientprocedure. PBMC were cultured directly or cryopreserved in RPMI-1640medium (PAA, Coelbe, Germany) containing 20% fetal bovine serum (FBS)(Bio West, Nuaille, France) and 10% DMSO and stored in the gas phase ofliquid nitrogen for later use. The HLA class I and class II haplotypesof the blood donors were typed by the Transplantation and ImmunologyLaboratory at the Institute of Immunology and Serology, Heidelberg,Germany. Sampling and use of PBMC were in accordance with theInstitutional Review Board at DKFZ and the University of Heidelberg,Heidelberg, Germany.

Depletion of CD4+ T Cells from PBMC

PBMC were depleted of CD4+ T cells according to the manufacturer'sinstruction using anti-CD4 MicroBeads (Miltenyi Biotec, BergischGladbach, Germany). Purity was assessed by flow cytometry. PBMC wereeffectively (>98%) depleted of CD4+ cells.

T-Cell Culture and IFN-γ ELISPOT Assay

PBMC were used for CD4+ T cell depletion or cultured directly inRPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 100U/ml penicillin and 100 g/ml streptomycin (R10 medium). Theimmunogenicity of candidate HPV16-derived peptides was evaluated byELISPOT analysis of short-term PBMC cultures. Briefly, PBMC were seededat a density of 10⁶ cells/well in 24-well plates (Becton Dickinson (BD),Heidelberg, Germany) in 1 ml of R10 medium, in the presence or absenceof individual HPV 16-derived peptides with a final concentration of 20μg/ml. As a positive control, PBMC were cultured in the presence of thepositive peptide pool (4 g/ml) or 5 g/ml of phytohaemagglutinin (PHA;Sigma). On day 5, PBMC were harvested, and seeded in triplicate wells ata density of 10⁵ cells/well on Multiscreen-HA ELISPOT plates (Millipore,Darmstadt, Germany) coated with 2 μg/ml of anti-human IFN-γ antibody1-DIK (Mabtech AB, Nacka Strand, Sweden). Cells were incubated foranother 20 h at 37° C. The ELISPOT assay was developed according to themanufacturer's instructions (Mabtech AB). The number of spots wasanalyzed with an AID ELISPOT reader (AID Diagnostika, Strassberg,Germany). Specific spots were calculated by subtracting the mean numberof spots of the DMSO background control from the mean number of spots oftest wells. A peptide-specific T cell response was considered positivewhen the mean spot number for a given peptide was at least twofoldhigher than mean background spot number.

In experiments in which selected HPV-derived peptides were furtheranalyzed, the PBMC or depleted PBMC were cultured for 12 days. Briefly,PBMC or depleted PBMC were seeded at a density of 5×10⁵ to 1×10⁶cells/well in 24-well plates in 1 ml of R10 medium supplemented withrecombinant human interleukin-7 (rhIL-7, R&D Systems, Wiesbaden,Germany) at a final concentration of 330 U/ml per well. Cells wereincubated in the presence of either DMSO (background control), selectedHPV16-derived peptides (20 μg/ml) or the positive peptide pool (4μg/ml). On days 3 and 7, cell culture was supplemented with rhIL-2(PeproTech, Hamburg, Germany) at a final concentration 20 U/ml per welland the medium was replaced (no rhIL-2) on day 10. Cells wererestimulated on day 11 with the indicated peptides (20 μg/ml). On day12, cultures were analyzed for the presence of peptide-specific T cellsby IFN-γ ELISPOT assay as described above.

To block peptide-specific IFN-γ production, anti-HLA-DR antibody L243(BioLegend, Fell, Germany) or anti-HLA class I antibody W6/32 were addedto the cell cultures at a final concentration of 10 μg/ml for 30 minbefore peptide exposure.

IFN-γ Intracellular Cytokine Staining

PBMC stimulated for 11 days with peptide as described above, wereharvested, washed and incubated in the presence of either DMSO orselected HPV16-derived peptide (20 μl/ml). GolgiPlug (1/1000; BD) wasadded after 1 h for a further 12 h incubation period. The cells werewashed and blocked with PBS/10% FBS and next stained for the surfacemarkers CD3-APC, CD4-PE, CD8-FITC (BD). For intracellular staining,cells were fixed and permeabilized using Cytofix/Cytoperm solutionaccording to the manufacturer's instructions (BD). The cells were washedwith Perm/Wash buffer (BD) before staining with anti-human IFN-γeFluor450-conjugated antibody (eBioscience, Frankfurt, Germany). Afterfixation with 1% formaldehyde, stained cells were analyzed on a FACSCanto II cytometer (BD). Respective isotype controls were used in allexperiments. Data analysis was performed with FlowJo (TreeStar, Ashland,Oreg., USA). Identical gates were used for all samples. A response wasconsidered positive for peptide-specific T cells if the response wasgreater than 0.04% after subtraction of the mean DMSO background.

Cytokine Assays

The supernatants collected on day 3 of the PBMC cultured with or withoutselected HPV-derived peptides were subjected to cytokine magnetic beadassay Milliplex MAP (Millipore) according to the manufacturer'sinstructions. The read-out of the assay was performed on a LuminexMAGPIX instrument with xPONENT software (Luminex, Austin, Tex., USA). Inthis assay, the levels of IFN-γ, TNF-α, GM-CSF, IL-4, IL-5 and IL-2 weredetermined. A peptide-specific cytokine production was consideredpositive if (after subtraction of the mean DMSO control) the cytokineconcentration was over the cutoff value of 20 μg/ml (50 μg/ml forIFN-γ).

Phenotypic CD4+ T Cell Analysis by Flow Cytometry

PBMC stimulated for 11 days with peptide as described above, wereharvested, washed and incubated in the presence of either DMSO orrelevant HPV-derived peptide (20 l/ml). GolgiPlug (1/1000; BD) was addedafter 1 h for a further 12 h incubation period. The cells were washedand blocked with PBS/10% FBS and next stained for the surface markersCD4-FITC, CD25-APC (BD). For intracellular staining, cells were fixedand permeabilized using Transcription Factor FIX/Perm Buffer accordingto the manufacturer's instructions (BD). The cells were washed withPerm/Wash buffer (BD) before staining with anti-human FoxP3PE-conjugated antibody (BD). After fixation with 1% formaldehyde,stained cells were analyzed on a FACS Canto II cytometer (BD).

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 5 Software Inc.Two-tailed Student's t-test with Welch's correction was used to analyzethe data. P values ≤0.05 were considered significant.

Results

Prediction and Selection of Candidate Peptides Derived from the HPV16E2, E5, E6 and E7 Proteins with High Binding Potency for Multiple HLA-DRMolecules

The inventors' study was designed to identify epitopes binding tomultiple HLA-DR alleles, derived from HPV16 early proteins E2, E5, E6and E7. Therefore the inventors first analyzed the amino acid sequenceof the indicated HPV16 proteins using two epitope prediction algorithms(Rammensee H, et al. 1999. SYFPEITHI: database for MHC ligands andpeptide motifs. Immunogenetics 50: 213-9; Nielsen M, et al. 2010.NetMHCIIpan-2.0—Improved pan-specific HLA-DR predictions using a novelconcurrent alignment and weight optimization training procedure.Immunome Res 6: 9; Nielsen M, Lund O. 2009. NN-align. An artificialneural network-based alignment algorithm for MHC class II peptidebinding prediction. BMC Bioinformatics 10: 296). 144 peptides for E6, 84peptides for E7, 351 peptides for E2 and 69 peptides for E5 binding toeleven different HLA-DR molecules were identified. To select promiscuousCD4+ T cell epitopes, for each protein, the 15-mer candidate peptideswere ranked according to their binding affinity to multiple HLA-DRalleles, excluding those harboring more than two cystein residues tominimize complexities resulting from intramolecular or intermoleculardisulfide bond formation. Six candidate pep-peptides of E6 and E7 aswell as eleven and eight candidate peptides from E2 and E5,respectively, which were predicted as binders by any one of theprediction algorithms and bound to at least 5 different HLA-DR molecules(Table 1) were synthesized and used for subsequent assays. As shown inTable 1, predictions were not completely consistent between the twoalgorithms.

Evaluation of HPV16 Peptide-Specific T Cell Immunity in Healthy Subjects

To investigate whether the predicted candidate peptides were able tostimulate T cells from a cohort of healthy donors, the inventors used a6-day IFN-γ ELISPOT assay. Frequencies of reactive T cells to eachcandidate peptide and the number of positive responders are reported forE6- and E7-derived peptides in Table 2, and for E2- and E5-derivedpeptides in Table 3. T cells isolated from the majority of healthysubjects showed reactivity against one or more of the HPV16-derivedcandidate peptides, after a single round of in vitro stimulation. Thepeptides E6/54-68 (30% response rate of tested healthy subjects),E6/74-88 (45%), E7/12-26 (40%), E7/64-78 (40%), E2/76-90 (30%),E2/99-113 (35%), E2/156-170 (40%), E5/33-48 (50%), E5/54-69 (55%), andE5/67-81 (60%) were found to be the most potent epitopes in this cohortof individuals expressing a wide variety of HLA-DR molecules. To furthercharacterize the immune potency of these ten selected peptides, PBMCfrom an independent set of 13 healthy subjects were analyzed in a 12 dayculture expansion of preexisting memory T cells. Using this approach,even higher frequencies of IFN-γ producing T cells against the selectedHPV16-derived peptides could be observed (FIG. 1). For each peptide ahigh response rate was observed, with 92% cumulative response rate(against all selected peptides) in the whole cohort of tested healthydonors. The five peptides showing the highest response rates and averagespot numbers (E2/156-170, E5/33-48, E6/74-88, E7/12-26 and E7/64-78)were chosen for further analysis.

Peptide Reactivity is Abrogated in the Presence of Anti-HLA-DRAntibodies

To determine whether the selected HPV16-derived peptides wereHLA-DR-restricted, PBMC were incubated with the HLA-DR blocking antibodyL243 before peptide exposure and then subjected to IFN-γ ELISPOT assay.Similarly, PBMC cultures were incubated with the pan-specific HLA classI blocking antibody W6/32. Two subjects were analyzed in this assay. Asshown in FIG. 2, peptide-specific T cell responses were abrogated in allcases under anti-HLA-DR antibody treatment, indicating that the epitopesare truly HLA-DR-restricted. However, the reactivity of the E5/33-48peptide was partially reduced when PBMC from subject HD54 were culturedwith the anti-panHLA-I antibody W6/32 (FIG. 2A). This was not observedwith this peptide with PBMC from HD55 (FIG. 2B). Further, blockingactivity of the anti-panHLA-I antibody was found in PBMC cultures ofboth subjects stimulated with E7/64-78 peptide, and withE6/74-88-stimulated PBMC from subject HD55. These results indicate thatthe 15-mer peptides E5/33-48, E6/74-88 and E7/64-78 are harboringepitopes restricted by some HLA class I molecules.

The Induced HPV16 Peptide-Specific T Cell Immunity is CD4+ TCell-Mediated

To verify whether the observed peptide-specific T cell responses areindeed mediated by CD4+ T cells, the inventors tested the five selectedpeptides in an IFN-γ intracellular cytokine secretion (ICS) assay in 7healthy subjects. Even if HPV16 peptide-specific T cell reactivitiesdetected by this assay were generally low, a predominance of CD4+ Tcells compared to CD8+ T cells could be observed (FIG. 3A). Thisdifference was found to be significant for E2/156-170 (P=0.013),E6/74-88 (P=0.01), E7/64-78 (P=0.05), but not for E5/33-48 (P=0.14) andE7/12-26 (P=0.10). This approach allowed us to assess T cell subsetstaking part in immune response against the selected HPV16 peptides.However, the inventors conclude that the ICS method is not sensitiveenough to assess preexisting HPV responses in healthy subjects.

To independently verify these findings with another method, PBMC weredepleted of CD4+ T cells (and, if sufficient PBMC were available from adonor, also of CD8+ T cells) prior to in vitro peptide stimulation andsubjected to ELISPOT analysis. The same five selected peptides wereanalyzed in an independent set of twelve subjects for this assay. CD4+ Tcell-depletion resulted in significant loss of peptide reactivity(P≤0.05) in the majority of tested subjects (FIG. 3B), again showingthat the peptide-specific immune responses are mediated by CD4+ T cells.CD8+ T cell-depletion did not affect peptide-specific IFN-γ production(FIG. 3C). However, reactivity of the E7/64-78 peptide was not abolishedin CD4+ T cell-depleted PBMC from subject HD24, indicating some CD8+ Tcell reactivity to this peptide in this donor. Taken together, thesedata provide strong evidence that the selected HPV16-derived peptidesare able to induce CD4+ T cell-mediated immunity.

Peptide-Specific CD4+ T Cells have a Th1 Phenotype

In order to investigate the functional properties of peptide-specificCD4+ T cells, the inventors assessed the Th1/Th2 cytokine profile.Analysis of supernatants of T cell cultures from HD23 for presence ofIFN-γ, TNF-α, GM-CSF, IL-4, IL-5 and IL-2 showed predomi-predominantsecretion of Th1 effector cytokines in response to the selectedHPV16-derived peptides (FIG. 4A). High levels of IFN-γ, TNF-α (exceptfor peptide E5/33-48) and GM-CSF were detected, whereas only weak or noproduction of IL-4 and IL-5 was observed. Interestingly, IL-2 was notdetected in these T cell cultures, which is probably due to itsconsumption by the proliferating T cells (this cytokine was not providedduring the PBMC culture). Furthermore, the inventors tested in fivesubjects whether the observed CD4+ T cell-mediated immunity in thepeptide-stimulated T cell cultures contains regulatory T cells (Treg).To do so, the inventors determined the frequency of peptide-specificCD4+/CD25+/FoxP3+ T cells after 12 days of peptide stimulation. Onlyminor populations of peptide-specific CD4+/CD25+/FoxP3+ T cells werefound in PBMC cultures after stimulation with the selected HPV16-derivedpeptides (FIG. 4B, representative results of one donor—HD23—are shown).

TABLE 1Candidate pan-HLA-DR binding peptides derived from the HPV16 E2, E5, E6 and E7 proteins SYFPEITHI High  Other NetMHCII Peptide scoring scoringHigh affinity Other binding (protein/ Sequence HLA-DR HLA-DRbinding HLA- HLA-DR position) (SEQ ID NO:) alleles^(a) alleles^(b)DR alleles^(c) alleles^(d) E6/16-30 PRKLPQLCTELQTTI DRB1*01,  DRB1*03,  DRB1*01, *04,  (SEQ ID NO: 1) *04 *07, *11,  DRB4 *15 E6/42-56QQLLRREVYDFAFRD DRB1*04 DRB1*01,   DRB1*01, *03,  (SEQ ID NO: 2)*03, *07,  *04, *15, DRB3,  *11, *15 DRB4, DRB5 E6/54-68 FRDLCIVYRDGNPYADRB1*04,  DRB1*01,   DRB1*01, *04,  (SEQ ID NO: 3) *11 *03, *15*11, *13, *15,  DRB3, DRB5 E6/74-88 LKFYSKISEYRHYCY DRB1*07 DRB1*01,  DRB1*01, *15,  DRB1*04, *07,  (SEQ ID NO: 4) *04, *11 DRB5 *09, *11E6/92-106 GTTLEQQYNKPLCDL DRB1*01,   DRB1*15 DRB1*01, *13, (SEQ ID NO: 5) *03, *04,  DRB4 *07 E6/129-143 KQRFHNIRGRWTGR DRB1*01, DRB1*07,   DRB1*01, *04,   C *04 *11, *15 *07, *09, *11, (SEQ ID NO: 6)*15, DRB5 E7/12-26 MLDLQPETTDLYCYE DRB1*03,   DRB1*01,  DRB1*01, *03(SEQ ID NO: 7) *04, *07 *15 E7/63-77 STLRLCVQSTHVDIR DRB1*01 DRB1*04, DRB1*01, *07,  DRB1*03, *04,  (SEQ ID NO: 8) *07 DRB4 *09, *11, *13,*15, DRB5 E7/64-78 TLRLCVQSTHVDIRT DRB1*01 DRB1*03,   DRB1*01, *07, DRB1*03, *04,  (SEQ ID NO: 9) *04, *07,  DRB4 *09, *11, *13, *15*15, DRB5 E7/71-85 STHVDIRTLEDLLMG DRB1*01,   DRB1*01, *03, (SEQ ID NO: 10) *03, *07,  *04, *07, DRB4 *15 E7/73-87 HVDIRTLEDLLMGTLDRB1*01,   DRB1*03,  DRB1*01 DRB1*03, *04, (SEQ ID NO: 11) *04, *07, *11  DRB4 *15 E7/76-90 IRTLEDLLMGTLGIV DRB1*01,  DRB1*03,   DRB1*01DRB1*04, *07,  (SEQ ID NO: 12) *04 *07, *11,  DRB4 *15 E2/27-42RDHIDYWKIIMRLEC DRB1*04,  DRB1*01,   DRB1*11 DRB1*01, *07,  A *15*03, *07,  *15, DRB4 (SEQ ID NO: 13) *11 E2/51-65 FKHINHQVVPTLAVSDRB1*01 DRB1*03,   DRB1*01, *07 DRB1*04, *09,  (SEQ ID NO: 14)*04, *07,  *11, *13, *15, *15 DRB4, DRB5 E2/59-73 VPTLAVSKNKALQAIDRB1*03,   DRB1*01,  DRB1*01, *13 DRB1*03, *07,  (SEQ ID NO: 15)*04, *07 *15 *09, *11,  DRB4, DRB5 E2/76-90 QLTLETIYNSQYSNE DRB1*03,  DRB1*01,   DRB1*01, *04,  (SEQ ID NO: 16) *04, *07 *11, *15 *15E2/99-113 LEVYLTAPTGCIKKH DRB1*01,   DRB1*11 DRB1*01, *07, DRB1*04, *09,  (SEQ ID NO: 17) *04, *07 DRB5 *11, *15, DRB3 E2/156-170GLYYVHEGIRTYFVQ DRB1*01,   DRB1*01, *07,  DRB1*03, *04,  (SEQ ID NO: 18)*03, *04,  *15, DRB3,  *09, *11, DRB4 *07, *11 DRB5 E2/208-223PEIIRQHLANHPAAT DRB1*01 DRB1*03,   DRB1*01, DRB4 DRB1*04, *07, (SEQ ID NO: 19) *04, *11,  *09, *11, *13, *15 *15, DRB5 E2/267-282ILTAFNSSHKGRINC DRB1*07, *09, DRB1*01, *04,  (SEQ ID NO: 20)  DRB5*11, *13, *15 E2/288-302 IVHLKGDANTLKCLR DRB1*03,  DRB1*01,   DRB1*03DRB1*01, *04,  (SEQ ID NO: 21) *04 *07, *15 DRB3, DRB5 E2/300-314CLRYRFKKHCTLYTA DRB1*01 DRB1*03,   DRB1*01, *07,  DRB1*04, *09, (SEQ ID NO: 22) *04, *07,  *11, *15 DRB3, DRB5 *11 E2/344-359DQFLSQVKIPKTITV DRB1*04 DRB1*01,   DRB1*01, *11,  DRB1*04, *07, (SEQ ID NO: 23) *03, *11,  DRB5 *09, *13, *15,  *15 DRB4 E5/1-15MTNLDTASTTLLACF DRB1*01,   DRB1*15 DRB1*01, *07 DRB1*03, *04, (SEQ ID NO: 24) *03, *04,  *09, *13 *07 E5/30-45 RPLLLSVSTYTSLILDRB1*01,  DRB1*03,   DRB1*01, *04,  DRB1*03, *09,  (SEQ ID NO: 25) *04*07, *11,  *07, *15 *11, *13, DRB4,  *15 DRB5 E5/33-48 LLSVSTYTSLILLVLDRB1*01,   DRB1*03 DRB1*01, *07,  DRB1*04, *09,  (SEQ ID NO: 26)*04, *07,  *15 *11, *15 E5/45-60 LVLVLW1TAASAFRC DRB1*01,  DRB1*03,  DRB1*01, *07,  DRB1*04, *11,  (SEQ ID NO: 27) *04 *07, *11,  *09, DRB5*13, *15, DRB3 *15 E5/54-69 ASAFRCFIVYIVFVY DRB1*01,   DRB1*07, DRB1*01, *11,  (SEQ ID NO: 28) *04, *15 *11 *15 E5/60-74 FIVYIVEVYIPLFLIDRB1*04,  DRB1*01,   DRB1*01, *07,  (SEQ ID NO: 29) *15 *07, *11*15, DRB3 E5/67-81 VYIPLFLIHTHARFL DRB1*11 DRB1*01,  DRB1*01, *04, DRB1*03, *09,  (SEQ ID NO: 30) *04 *07, *11, *13, DRB3, DRB4 *15, DRB5E5/69-84 IPLFLIHTHARFLIT DRB1*01,  DRB1*03,   DRB1*01, *03, DRB1*09, DRB3,  (SEQ ID NO: 31) *11 *04, *07 *04, *07, *11,  DRB4*13, *15, DRB5 ^(a)Threshold for good binding peptides: score ≥ 20.^(b)Threshold for other binding peptides: score 19 ≤ 10. ^(c)Thresholdfor good binding peptides: IC₅₀ ≤ 60 nM. ^(d)Threshold for other bindingpeptides IC₅₀: 60 ≤ 1000 nM.

TABLE 2 HPV16 E6- and E7-specific T-cell responses in healthy donors asmeasured by IFN-γ ELISPOT HLA- HPV16 E6 peptides HPV16 E7 peptides DRB1E6/129- E7/12- E7/63- E7/64- E7/71- E7/73- Donor genotype E6/16-30E6/42-56 E6/54-68 E6/74-88 E6/92-106 143 26 77 78 85 87 E7/76-90 HD1*04, —  55^(a) 12 25 35 — 88 128 58 98 88 82 192 HD2 *03, *11 — 27 — — —13 13 — — — — — HD3 *03, *04 187 260 233 183 63 113 80 80 133 177 180323 HD4 *04, *16  17 — 30 — 7 7 — 27 — — — — HD5 *01, *16  2 288 392 528— 122 28 205 185 — — — HD6 *03, *13 — 50 130 220 7 97 567 3 63 13 63 20HD7 *08, *11 — — — — 7 — 3 3 — 60 7 — HD8 *13, — — — — — — — — 63 — — —— HD9 *01, *07  2 2 2 35 18 2 — 18 5 — — — HD10 *04, *13  85 15 2 142 1845 68 78 245 58 115 48 HD11 *01, *04 250 163 77 73 70 207 347 113 133 43— 117 HD12 *03, *11 263 73 250 393 243 83 223 447 270 350 190 223 HD13*11, *14 125 105 108 222 168 135 258 68 265 15 72 38 HD14 *07, *13 — 14333 213 87 37 190 140 227 127 40 113 HD15 *01, *08  20 37 63 40 13 10 6387 130 237 67 73 HD16 *07, *13  45 195 18 22 45 25 192 172 98 328 72 22HD17 *01, *15 142 282 132 332 235 458 342 242 812 438 298 278 HD18 *08,*13  5 162 — 85 — 5 258 228 215 — 48 98 HD19 *01, *15 518 162 362 168108 — 355 315 418 215 202 82 HD20 *07, *15 175 85 75 95 115 118 318 128108 192 165 95 Positive 5 (25%) 5 (25%) 6 (30%) 9 (45%) 2 (10%) 5 (25%)8 (40%) 5 (25%) 8 (40%) 6 (30%) 4 (20%) 3 (15%) responders (%)^(a)Number of peptide-specific memory T cells per 10⁶ PBMC. Datarepresent the mean result from triplicate wells after backgroundsubtraction. A dash indicates that no specific T cell reactivity wasdetected. Responses are considered positive (bold) if they were at leasttwofold higher than the background.

TABLE 3 HPV16 E2- and E5-specific T-cell responses in healthy donors asmeasured by IFN-γ ELISPOT HLA- HPV16 E2 peptides DRB1 E2/267- E2/288-E2/300- E2/344- Donor genotype E2/27-42 E2/51-65 E2/59-73 E2/76-90E2/99-113 E2/156-170 E2/208-223 282 302 314 359 HD21 *07, *13 123 ^(a) 7 47 50 147  13 13  7 13  7 40 HD22 *04, *15 —  3  3  7 27  7 — 10  3 — 3 HD23 *11, *13 — — — — — 10 — 13 20 13 23 HD24 *09, *12  3  7  7 — 2010 17 10  3 10 — HD25 *03, *15 170 — —  3 110  — — — — — — HD26 *04, *16— — — — 10 23 17 — — 30 13 HD27 *01, *11  10 57 87 60 133  53 40 87  377 73 HD28 *11, *15  27 27 — 223  — 493  73 17 57  7 10 HD29 *04, *16 17 23 — 57 33 87  3 13 — 143   7 HD30 *07, *11 — 10 — —  3  3  3 — —  713 HD31 *07, *11 — — —  3 50 77 47 23 27 20  3 HD32 *01, *16 — —  3  740 40  7  3  3 — 10 HD33 *04, *07 150 40 173  120  73 617  93 — 270 123  103  HD34 *04, — — — 13  3 — —  7 10 — — 13 HD35 *11, —  3 17 33 20— —  7 —  7 17 30 HD36 *04, —  47 — — — 47 403  — — 13 127  123  HD37*04, *07  47 10 167  307  83 167  17 40 77 123  43 HD38 *13, *15 100 1030 33 350  47 53 23  7 27 37 HD39 *04, *13  10 17 27 33 13 13 —  3 — 83107  HD40 *03, *04 — 10 37 17  3 23 —  3 — 20  3 Positive  3  2  4  6  7 8  3  2  2  5  3 responders (15%) (10%) (20%) (30%) (35)% (40%) (15%)(10%) (10%) (25%) (15%) (%) HLA- DRB1 HPV16 E5 peptides Donor genotypeE5/1-15 E5/30-45 E5/33-48 E5/45-60 E5/54-69 E5/60-74 E5/67-81 E5/69-84HD21 *07, *13  7 — 67 20 153  97 40  3 HD22 *04, *15  3  3 17 13  7 220  7 10 HD23 *11, *13 —  3 — 43 193  200  —  3 HD24 *09, *12 —  7 23 — 17433   3  3 HD25 *03, *15 13 70 207  43 7 17 27 23 HD26 *04, *16 —  7 —77 50 33 100  — HD27 *01, *11 223  247  73 97 140  200  133  13 HD28*11, *15 270  60 37 250  143  147  180  63 HD29 *04, *16  3 — 13 — 10 4060 113  HD30 *07, *11 — — —  3  7 27 13 — HD31 *07, *11 13 73  7  3 80107  — — HD32 *01, *16 30 47 — — 33 13 —  3 HD33 *04, *07 283  130  500 407  367  27 73 110  HD34 *04, — — 30 30  7 40 43 — — HD35 *11, — 10 10170  20 157  207  50 20 HD36 *04, — — 80 77 — 27 40 17 — HD37 *04, *07 —183  50 297  80 127  93 17 HD38 *13, *15 53 173  160  247  280  183 227  50 HD39 *04, *13  3 10 27 10 73 47 23 — HD40 *03, *04 30 17 20 2053 27 20 23 Positive  4  5 10  6 11 12  7  3 responders (20%) (25%)(50%) (30%) (55%) (60%) (35%) (15%) (%) ^(a)Number of peptide-specificmemory T cells per 10⁶ PBMC. Data represent the mean result fromtriplicate wells after background subtraction. A dash indicates that nospecific T cell reactivity was detected. Responses are consideredpositive (bold) if they were at least twofold higher than thebackground.

TABLE 4 Comparison of the predicted HLA-DRB1 alleles with theexperimentally tested HLA-DRB1 molecules presenting selectedHPV16-derived peptides Peptide HLA-DRB1 molecules (protein/ predicted tobind a HLA-DRB1 genotype of position) given peptide responders E6/54-68*01, *03, *04, *11, *13, *03/*04; *01/*16; *03/*13; *15 *03/*11;*01/*08; *01/*15 E6/74-88 *01, *04, *07, *09, *11, *03/*04; *01/*16;*03/*13; *15 *01/*07; *04/*13; *03/*11; *07/*13; *01/*08; *01/*15E7/12-26 *01, *03, *04, *07, *15 *03/*04; *03/*13; *01/*04; *03/*11;*01/*08; *01/*15; *07/*15 E7/64-78 *01, *03, *04, *07, *09, *03/*04;*03/*13; *04/*13; *11, *13, *15 *03/*11; *07/*13; *01/*08; *01/*15E7/71-85 *01, *03, *04, *07, *15 *03/*04; *08/*11; *03/*11; *01/*08;*07/*13; *01/*15 E2/76-90 *01, *03, *04, *07, *11, *07/*13; *01/*11;*11/*15; *15 *04/*16; *04/*07; *04/*13 E2/99-113 *01, *04, *07, *09,*11, *07/*13; *09/*12; *03/*15; *15 *01/*11; *04/*07; *13/*15 E2/156-170*01, *03, *04, *07, *09, *09/*12; *01/*11; *11/*15; *11, *15 *04/*16;*04/*07; *04/—; *13/*15 E5/33-48 *01, *03, *04, *07, *09, *07/*13;*09/*12; *03/*15; *11, *15 *01/*11; *04/*07; *13/*15; *04/*13 E5/45-60*01, *03, *04, *07, *09, *11/*13; *01/*11; *11/*15; *11, *13, *15*04/*07; *13/*15 E5/54-69 *01, *04, *07, *11, *15 *07/*13; *11/*13;*09/*12; *01/*11; *11/*15; *04/*07; *11/—; *13/*15; *04/*13; *03/*04E5/60-74 *01, *04, *07, *11, *15 *07/*13; *04/*15; *11/*13; *09/*12;*01/*11; *11/*15; *07/*11; *04/—; *11/—; *04/*07; *13/*15; *04/*13E5/67-81 *01, *03, *04, *07, *09, *04/*16; *01/*11; *11/*15; *11, *13,*15 *04/*07; *13/*15; *04/*13 Peptides in bold: selected for detailedanalysis. HLA-DRB1 molecules in bold: predicted as high affinity bindingor scoring.

1. A peptide comprising a sequence according to SEQ ID NO: 4 or avariant thereof that is at least 80% homologous to SEQ ID NO: 4, whereinsaid peptide, or one or more parts thereof, has the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-II or-I, and wherein said peptide has a length of 15 to 50 amino acids. 2.The peptide according to claim 1, wherein said peptide or variant hasfrom 15 to 30 amino acids.
 3. The peptide according to claim 1, whereinsaid peptide consists of the amino acid sequence according to SEQ ID NO:4.
 4. The peptide according to claim 1, wherein said peptide has anIC₅₀≤60 nM.
 5. A pharmaceutical composition comprising the peptideaccording to claim 1, and optionally at least one additional peptideconsisting of an amino acid sequence selected from the group of SEQ IDNOs: 26, 9, 7, and
 18. 6. The peptide according to claim 1, wherein saidpeptide is part of a fusion protein or other fusion molecule.
 7. Anucleic acid encoding the peptide according to claim
 1. 8. An expressionvector capable of expressing the nucleic acid according to claim
 7. 9. Ahost cell comprising a nucleic acid encoding the peptide of claim 1,wherein said host cell is an antigen presenting cell and presents thepeptide according to claim
 1. 10. A method for producing the peptideaccording to claim 1, said method comprising synthesizing said peptide,or culturing a host cell comprising a nucleic acid encoding the peptideof claim 1, wherein said host cell is an antigen presenting cell andpresents the peptide according to claim 1, and isolating said peptidefrom said host cell or a culture medium thereof.
 11. A method forproducing activated T cells, the method comprising contacting T_(H)cells with antigen loaded human class I or II MHC molecules expressed onthe surface of suitable antigen-presenting cells for a period of timesufficient to activate said T cells in an antigen specific manner,wherein said antigen is a peptide according to claim
 1. 12. An activatedT cell, produced by the method according to claim 11, wherein said Tcell selectively recognizes cells that express an HPV polypeptidecomprising a sequence according to SEQ ID NO: 4 or a variant thereofthat is at least 80% homologous to SEQ ID NO: 4, wherein said peptide,or one or more parts thereof, have the ability to bind to a molecule ofthe human major histocompatibility complex (MHC) class-II or -I, andwherein said peptide has a length of from 15 to 50 amino acids.
 13. Apharmaceutical preparation, comprising at least one of: a nucleic acidencoding the peptide according to claim 1; a host cell comprising anucleic acid encoding the peptide of claim 1, wherein said host cell isan antigen presenting cell and presents the peptide according to claim1; an activated T cell, wherein said T cell selectively recognizes cellsthat express an HPV polypeptide comprising a sequence according to SEQID NO: 4 or a variant thereof that is at least 80% homologous to SEQ IDNO: 4, wherein said peptide, or one or more parts thereof, has theability to bind to a molecule of the human major histocompatibilitycomplex (MHC) class-II or -I, and wherein said peptide has from 15 to 50amino acids; and wherein said preparation further comprises apharmaceutically acceptable excipient.
 14. A method for treating HPVinfection, HPV-related premalignancies and/or malignancies comprisingadministering the pharmaceutical preparation according to claim 13 to apatient in need of said treatment.
 15. The method for treating HPVinfection, HPV-related premalignancies and/or malignancies according toclaim 14, wherein said treatment is MHC-I and/or MHC-II peptidepresentation dependent.
 16. The peptide according to claim 1, whereinsaid peptide or variant has from 15 to 20 amino acids.